1. Field of the Invention
The present invention is a method for mapping nucleic acid molecules. More specifically, it is a method for determining where one oligonucleotide hybridizes to another oligonucleotide.
2. Description of Related Disclosures
It is sometimes useful to determine where one oligonucleotide sequence resides within another. One familiar example of this is the preparation of a conventional restriction map, a technique reviewed by Nathans and Smith. If a circular plasmid is digested with one or more restriction enzymes which are known to cut, in combination, two or more times within the plasmid, a restriction map can sometimes be prepared to show where the restriction enzymes cut the plasmid and, equivalently, how the fragments produced by the digestion are arranged within the plasmid. Once a restriction map has been prepared it may then be said that a certain fragment resides at a certain location within the plasmid.
The conventional preparation of such a restriction map involves performing multiple restriction digests using individual enzymes and combinations of enzymes. The digestions are followed by an analysis of the lengths of the fragments produced. This analysis involves considering all of the potential permutations that the fragments might embody and becomes factorially more difficult as the number of fragments increases. In many cases an unambiguous map cannot be prepared. Restriction maps also require that more than one restriction enzyme be mapped at a time. Moreover, conventional mapping procedures cannot use the data from a single enzyme digestion to prepare a map of that enzyme.
Hybridization mapping avoids several of the problems associated with conventional restriction mapping. It does not require that multiple restriction digests be performed to create a map. Nor does it involve the ambiguity and potential failure entailed in determining the correct permutation of fragments produced by multiple digests.
Another method for locating one oligonucleotide within another is the method of Berk and Sharp for locating the portion of an mRNA transcript within a DNA clone which is suspected of having served as a transcription template for the transcript. The Berk-Sharp method involves preparing multiple restriction maps of the DNA clone by the method described above, determining the complete size of the transcripts of interest and then determining the sizes of the hybrids formed between the transcripts and the different restriction fragments from the different restriction digests. The determination of size information is done by completely digesting away the single-stranded regions of the transcript-restriction fragment hybrids with S1 nuclease and then sizing the double-stranded hybrids on an electrophoretic gel. The size information can then be analyzed to locate the transcription location on the full restriction map of the large DNA clone.
Hybridization mapping does not require that either the target or probe oligonucleotides be restriction mapped beforehand, as does the Berk-Sharp method. The Berk-Sharp method also requires that a significant amount of analysis be performed on the data produced and often requires that additional restriction maps of the DNA clone be prepared by other methods to produce an unambiguous result. Another deficiency of the Berk-Sharp method is that it is designed to only work with RNA-DNA hybrids whereas hybridization mapping will work with DNA-DNA hybrids as well as RNA-DNA hybrids.
Another situation in which it is useful to learn where one oligonucleotide resides within another is when sequencing a very long molecule such as a chromosome. One current method of long-molecule sequencing is to randomly divide the long molecule into multiple small overlapping fragments, sequence the fragments, and then use a computer to determine the regions of overlap. This "shotgun" sequencing technique results in the long molecule being sequenced multiple times and there are often problems in obtaining the last few fragments needed to produce a complete sequence of the molecule.